Injectable contraceptive vaccine and method

ABSTRACT

An injectable contraceptive vaccine, a method of preparing said vaccine and the method of preventing conception in mammals by use of said vaccine, is disclosed. A vaccine which stimulates the production of antibodies directed against a hormone essential to the reproductive cycle of a mammal is prepared by coupling such a hormone to a carrier to form a hormone-carrier conjugate. Conception in mammals is prevented by injection of such vaccine during the proper period of the reproductive cycle of said mammal. 
     Specifically described is the vaccine prepared by coupling a hormone, such as estradiol-17 beta, with a protein and its use to sterilize a female of the canine species.

This is a continuation of application Ser. No. 715,163 filed Aug. 17,1976 now abandoned.

SUMMARY OF INVENTION

This invention relates to a contraceptive vaccine, to a method of itspreparation and to a process for the prevention of conception in femalemammals by the use of such vaccine.

Particularly, the invention relates to a contraceptive vaccine whichstimulates the production of antibodies to an estrogen, which estrogenis essential to the reproductive cycle of a female mammal, and whichantibodies have been produced by the immunological system of the femalemammal.

Still more particularly, the invention comprises a contraceptive vaccinewhich stimulates the production of antibodies against the estrogen,estradiol-17 beta (E₂), when injected into a female mammal such that anantibody level of at least 70 PEA units per ml blood serum exist in suchfemale at the time of expected estrus.

GENERAL DESCRIPTION OF INVENTION

There exists today a considerable interest on the part of thoseconcerned with animal husbandry, and others, in the prevention ofconception, at least for some period of time, in most mammals, whetherthey be primate, canine, feline, bovine, ovine porcine, equine, and thelike. Consequently, there have been many methods derived to accomplishthis end, all of which are undesirable in one respect or another. Forexample, clinical surgery (castration, ovari-hysterectomy) has itsobvious faults. Mechanical contraception is likewise undesirable forvarious reasons and chemotherapeutic agents are not only unreliable, butalso many times induce undesirable side effects in many mammals.

It has long been known that female mammals produce estrogen which isnecessary for the manifestation of estrus, ovulation and implantation.If the mammalian body were to produce its own antibodies against such arequisite estrogen, fertility would not exist.

The natural occurring estrogen molecule in the mammalian body isnonantigenic and therefore will not stimulate the production ofantibodies of said estrogen in the body of said mammals.

The present invention involves producing a vaccine which, when injectedinto a mammal, will stimulate the production of antibodies against arequisite estrogen. This is accomplished by first making an antigen bycoupling an essential estrogen to a carrier and injecting such antigeninto the mammal to cause sterilization of, or prevent conception in,said mammal.

Of particular interest, and contemplated in one preferred embodiment ofthis invention, is a contraceptive vaccine which stimulates theproduction of antibodies against the estrogen, estradiol-17 beta (E₂),which vaccine has been found useful to control canine reproduction.

It has been found that if the contraceptive vaccine is given, one canexpect a contraceptive effect.

The preparation of the novel contraceptive vaccine of this invention maybe generally described as follows.

The vaccine of the present invention comprises a steroid-carrierconjugate which has been found to be effective in producing specificantibodies against the steroid. For example, an estrogen such asestradiol-17 beta(E₂) is rendered antigenic by covalent attachment to acarrier. The carrier selected for use in the production of the vaccinemay be, for example, any protein or polypeptide, which, when combinedwith the said estrogen molecule, will render the said steroid-carrierconjugate antigenic.

In one specific embodiment of the inventive concept, the conjugation ofthe estradiol-17 beta (E₂) to the amino groups of the carrier used iseffected by preparing the hemisuccinate of the estrogen molecule, whichhemisuccinate is then coupled to the carrier via the carbodiimidereaction.

As is well known in the art, other estrogens may be rendered antigenicby coupling to other carriers by known chemical reactions. For example,estrogens such as estradiol-17 alpha, estrone, estriol, equilin,equilenin, and the like may be coupled to a carrier which, in itself,may be antigenic, or may be modified to become antigenic. Examples ofsuch carriers are Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin(BSA), Human Serum Albumin (HSA), and the like.

A derivative of the desired estrogen is prepared, for example, byesterification of the hydroxyl groups of the estrogen with succinicanhydride, forming the oxime derivatives of the ketone groups of theestrogen using (o-carboxymethyl) hydroxylamine, and the like, and thederivative is then coupled to the carrier by, for example, theSchotten-Baumann method, the mixed anhydride method, the carbodiimidecondensation, and the like. These techniques are well known to the art.See for example, Thorneycroft, I. H. (1970). Preparation andPurification of Antibodies to Steroids. Immunologic Methods in SteroidDetermination, Appleton-Century-Crofts, 63-86; Lindner, et al. 1972.Specificity of Antibodies to Ovarian Hormones in Relation to the Site ofAttachment of the Steroid Hapten to the Peptide Carrier. Steroids19:357; Mahajan, D. K., et al. 1972. Plasma 11-DeoxycortisolRadioimmunoassay for Metyrapone Tests. Steroids 20:609; Yellin, T. O.1972. Estradiol-17-Beta-Hemisuccinate: An Improved Procedure. J. LipidRes. 13:554; Lieberman, et al. 1959. Steroid-Protein Conjuates: TheirChemical, Immunochemical, and Endocrinological Properties. Rec. Prog.Hor. Res. 15:165.

The prior art has also described the use of various estrogen antibodiesin the investigation of mechanism of the reproductive cycles of mice,(Ferin, M., et al. 1968. Inactivation of the Biological Effects ofExogenous and Endogenous Estrogens by Antibodies to 17 Beta-Estradiol.Endocrinology 83:565), rats, (Ferin, M. A. et al. 1969. Effect ofAntibodies to 17 Beta-Estradiol and Progesterone on the Estrous Cycle ofthe Rat. Endocrinology 85:1070) and sheep, (Scaramuzzi, R. J. 1975.Inhibition of Oestrous Behaviour in Ewes by Passive Immunization AgainstOestradiol-17 Beta. J. Reprod. Fert. 42:145).

The female to be protected is injected with the desired amount ofvaccine, parenterally, preferably both intradermally andintramuscularly. After an initial injection, the female may be injectedperiodically in order to maintain a satisfactory antibody level, andthus maintain sterilization.

PREPARATION OF VACCINE

The vaccine of one preferred embodiment of this invention consists of asteroid-protein conjugate found to be effective in producing specificantibodies against the steroid (E₂) residue. Estradiol-17 beta (E₂) isrendered antigenic by covalent attachment to the protein Bovine SerumAlbumin (BSA). Coupling to epsilon-amino groups of the lysine residuesof BSA is affected via the 17-beta hemisuccinate by the use of thecarbodiimide reagent. The complete conjugation procedure employed is amodification of procedures employed by previous researchers.

1. Preparation of Estrogen Derivative

Five grams of E₂ (Calbiochem, San Diego, Cal.) and 25 microCuries (uCi)of ³ H-6,7-estradiol-17 beta (³ H-E₂) (New England Nuclear, Boston,Mass.) are added to a 500 ml round-bottom flask. Next, 250 ml benzenecontaining 1% pyridine and 15 gm succinic anhydride are added to thesame flask and the contents refluxed for 40 hours. The solvent is thenevaporated and the remaining residue is dissolved in 625 ml methanol. Tothe solution is added 100 ml of a 15% solution of sodium bicarbonate andthe contents are stirred overnight. The contents are filtered and anequal volume of water is added. The solution is then extracted threetimes with 200 ml portions of diethyl ether. The remaining aqueous phaseis adjusted to pH7 with 6 N HCL and poured into a mixture of 0.2 N HCLand crushed ice (1:1). The precipitate, estradiol-17 beta hemisuccinate(E₂ -HS), is collected by filtration, washed with water and dried invacuo at 37° C.

The general chemical scheme leading to the formation of theestradiol-hemisuccinate is as follows: ##STR1##

In order to subsequently determine the number of moles of E₂ which areattached to a mole or protein, the disintegrations per minute per mg E₂-HS (DPM/mg E₂ -HS) must be determined. To accomplish this, 45 to 55 mgof accurately weighed E₂ -HS is placed into a liquid scintillationcounting vial. To the vial is also added 0.1 ml water and 1 ml ofSoluene-350 (solubilizer, Packard Instrument Co., Downers Grove, Ill.)and the contents are allowed to stand until the residue is completelydigested. After this digestion, 10 ml of Dimulume (Scintillation media,Packard Instrument Co.) is added and the contents of the vial arecounted for radioactivity. The DPM/mg E₂ -HS is then determine basedupon counts per minute (CPM), scintillation counter efficiency andweight of E₂ -HS. The DPM/mg E₂ -HS is calculated in the followingmanner: Example: ##EQU1##

2. Conjugation of Estrogen Derivative to Carrier

Five grams of BSA (Sigma) is dissolved in 250 ml of water the the pH isadjusted to 7.8. With constant stirring, 2.5 gm of water-solublecarbodiimide [1-ethyl-3 (3-di-methyl amino propyl) - carbodiimide] isadded while maintaining the above pH dilute HCl. There is added dropwise50 ml of dimethyl formamide containing 2.5 gm E₂ -HS with constantstirring while maintaining the pH at 7.8 with 1 N NaOH. The reactionmixture is equilibrated at room temperature for 2 hours, after which anadditional 500 mg of the carbodiimide is added. After an additional 20hour equilibration, the mixture is dialyzed against water for 48 hours.The steroid-protein conjugate is precipitated by the addition of 90 mlacetone for each 10 ml of solution remaining in the dialysis bag. Theresidue is collected by filtration, washed with acetone and dried invacuo at 37° C. The general chemical scheme leading to the formation ofE₂ -BSA from E₂ -HS is as follows: ##STR2##

3. Testing of Vaccine

Ten to 15 mg of accurately weighed steroid-protein conjugate (E₂ -BSA)is placed into a liquid scintillation counting vial. To the vial isadded 0.1 ml water and 1 ml of Soluene-350 and the contents are allowedto stand until the residue is completely digested. After this digestion,10 ml of Dimulume is added and the contents of the vial are counted forradioactivity. The number of moles of E₂ which is attached to one moleof BSA is calculated in the following manner:

EXAMPLE:

1. molecular weight (M.W.) of BSA = 7 × 10⁴ gm/mole

2. DPM/mg E₂ -HS as determined under 1. above - 4500 DPM/mg E₂ -HS

3. m.w. of E₂ -HS = 372 gm/mole

4. 5300 DPM/10 mg of E₂ -BSA

Calculations: ##EQU2##

It has been found, and forms an important part of the inventive concept,that the E₂ -BSA antigen is most effective in producing specificantibodies against the steroid moiety when the number of E₂ -HSmolecules incorporated per molecule of BSA is between 15 and 25.

As was stated above, the vaccine of this invention is an E₂ proteinconjugate, prepared as described above.

Each dog to be protected receives an initial inoculation of 1 mg E₂ -BSAemulsified in 2 ml of adjuvant with a concentration of 0.5 mg/ml. Theadjuvant consists of an emulsion containing by volume:

50% Complete Freund's Adjuvant

50% water

One ml is injected into multi-intradermal sites. The other 1 ml isinjected into 2 intramuscular (I.M.) sites.

In order to determine whether the proper antibody levels are maintained,the following assay system has been performed. The serum is titered forantibodies against E₂ by the capacity of the serum to bind theradioactive form of E₂, ³ H-6,7-E₂ (³ H-E₂). Serial dilutions of theserum are carried out to obtain and end point binding of approximately50% of the ³ H-E₂ added. The dilution of serum that gives approximately50% binding is used to calculate the antibody level. Using the variousserum dilutions, a sigmoid curve is obtained and 50% binding fits intothe linear most portion of the curve. A charcoal suspension is used toseparate antibody-bound ³ H-E₂ from free ³ H-E₂. If one adds 10,000 cpmof ³ H-E₂ and after charcoal separation finds 5000 cpm of ³ H-E₂ boundto the antibody, one therefore achieves a 50% binding at a particularserum dilution.

In order to calculate the amount of ³ H-E₂ found in each tube perdilution, the mass of the original ³ H-E₂ added must be determined asshown in the following calculation.

EXAMPLE:

1. m.w. of ³ H-E₂ = 272.4 gm

2. Specific Activity of ³ H-E₂ = 49.3 Ci/m Mole

3. 2.22 × 10⁶ DPM per uCi

4. CPM ³ H-E₂ added per culture tube = 10,414

5. 1 gm = 1 × 10¹² picograms (pg)

6. Efficiency of counting = 46.6%

Therefore:

1. 49.3 Ci = 49.3 × 10⁶ uCi

2. mMole of ³ H-E₂ = 0.2724 gm

(49.3 × 10⁶ uCi) (2.22 × 10⁶ DPM/uCi) = 109.446 × 10¹² DPM/0.2724 gm##EQU3## Once the mass of the ³ H-E₂ has been determined, the bindingcapacity of the serum can be calculated as shown in Table I.

                  TABLE I                                                         ______________________________________                                        Binding Capacity of the Serum                                                                          pg                                                                    %       .sup.3 H-E.sub.2                                                                            pg                                                      .sup.3 H-E.sub.2                                                                      Bound         .sup.3 H-E.sub.2                       Serum            Bound   (%            Bound                                  Dilution         (CPM/   bound × per ml                                 (ml)     CPM     10,414) 55.6)  Factor serum                                  ______________________________________                                        1/100*   8346*   77,9*   44.2*  × 100                                                                           4,420*                                1/500*   8147*   76.0*   43.2   × 500                                                                          21,600*                                1/1000   7548    72.5    40.3   × 1000                                                                         40,300                                 1/2000   6182    59.4    33.0   × 2000                                                                         66,000                                 1/2500** 5086**  48.8**  27.1** 2 2500 68,000**                               1/4000   2845    27.3    15.2   × 4000                                                                         60,800                                 1/5000   2040    19.6    10.9   × 5000                                                                         54,500                                 1/10,000  797    7.7     4.3    × 10,000                                                                       43,000                                 ______________________________________                                         *Determined from another assay where added CPM = 10,718 and pg = 56.8         **Dilution coming nearest to 50% binding                                 

Calculations are based on the reading at the 1/2500 dilution since atthis dilution approximately 50% of the ³ H-E₂ is bound. Therefore, 1 mlof the serum has the ability to bind 68,000 pg of E₂ or 68,000 pgequivalent antibody (PEA) units.

TREATMENT OF FEMALE MAMMALS

All female dogs to be treated with the vaccine of this invention wereselected with known reproductive histories consisting of at least onepreviously recorded estrus period. The females were injected with either0.5 or 1.0 mg of the vaccine prepared as described above. Data obtainedfrom 15 treatment cycles representing 9 dogs is set out in Table IIbelow.

                                      TABLE II                                    __________________________________________________________________________    Active Immunization with Estradiol-17 Beta-                                   Bovine Serum Albumin (E.sub.2 -BSA) for                                       Sterilization in the Female Dog                                                                             D     E                                                          B            Duration                                                                            Anti-E.sub.2                                               Duration     from Pre-                                                                           Levels                                    Immunization     of Anti-                                                                            C      Treatment                                                                           at                                               Amount                                                                             A    E.sub.2 Levels                                                                      Theoretical                                                                          Estrus to                                                                           Actual                                       No. of                                                                            of E.sub.2 -                                                                       Injection                                                                          Above 70                                                                            Protection                                                                           Actual                                                                              Estrus                                    Dog                                                                              Injec-                                                                            E.sub.p BSA                                                                        Interval                                                                           PEA Units                                                                           Time (A+B)                                                                           Estrus                                                                              (PEA                                      No.                                                                              tions                                                                             (mg) (Mo.).sup.c                                                                        (Mo.) (Mo.)  (Mo.) Units)                                    __________________________________________________________________________    D09                                                                              1   1.0  3.25 3.00  6.25   12.25 <10                                       D11                                                                              1   0.5  3.25 3.00  6.25   8.25  40                                        D11.sup.a                                                                        1   1.0  2.25 2.00  4.25   5.75  <20                                       D06.sup.b                                                                        3   1.0  3.25 30.50+                                                                              33.75+ 33.75+.sup.e                                                                        --                                        3370                                                                             1   0.5  3.25 2.75  6.00   7.00  40                                        3370.sup.a                                                                       1   1.0  3.25 3.00  6.25   6.50  60                                        4974                                                                             1   1.0  3.25 1.25  4.50   7.50  40                                        5415                                                                             1   1.0  3.25 3.75  7.00   7.50  45                                        5415.sup.a                                                                       1   1.0  3.00 3.50  6.50   13.75 <10                                       5458                                                                             1   1.0  3.25 6.25  9.50   10.75 25                                        5458.sup.a                                                                       1   1.0  0.25 6.75+.sup.d                                                                         7.00+  7.00  408                                       5795                                                                             1   1.0  3.25 3.00  6.25   6.25  45                                        5795.sup.a                                                                       1   1.0  4.25 2.00  6.25   7.50  50                                        5634                                                                             1   1.0  6.00 0.50  6.50   8.75  <10                                       4974.sup.a                                                                       1   1.0  3.00 0.50  3.50   7.50  <10                                       __________________________________________________________________________     .sup.a Received E.sub.2BSA following the first post treament estrus           .sup.b Received a second and third injection of E.sub.2BSA 81/2 and 19        months, respectively, following the first injection.                          .sup.c Interval from pre-treatment estrus to injection                        .sup.d Antibody levels were not measured after estrus                         .sup.e This female has not returned to estrus at the time of this writing                                                                              

It has been found that the bitch produces at maximum 68.9± 11.0 pg ofestradiol-17 (E₂) per ml serum (Nett et al. 1975. Levels of LuteinizingHormone, Estradiol and Progesterone in Serum During The Estrous Cycleand Pregnancy in the Beagle Bitch. Proc. Soc. Exp. Biol. Med. 148:134).Therefore, if one administers an estrogen-protein conjugate andstimulates the production of antibodies against E₂ (anti-E₂) sufficientto bind in excess of 70 pg of E₂ per ml serum, one should achievesterilization of the female dog.

Preliminary studies indeed indicate evidence (Table II) to support theclaim that sterilization of the female dog can be brought about byactive immunization with administered estrogen-protein conjugate(vaccine).

It can be noted from Table II that with the exception of one treatmentcycle (No. 5458, second treatment cycle) at no time did estrus occurwhen the binding capacity of anti-E₂ exceeded approximately 70 pg of E₂per ml of serum. It can be assumed from Table II that if anti-E₂ levelswere maintained to bind 70 or more pg of E₂ per ml of serum, one couldobtain a more pronounced contraceptive effect.

If the claim that anti-E₂ levels capable of binding 70 or more pg of E₂per ml of serum will elicit a contraceptive response in female dogs isvalid, the duration from pre-treatment estrus to actual estrus followingtreatment (Column D) should exceed the theoretical protection time(Column C). This is indeed the case with the exception of one treatmentcycle (No. 5458, second treatment cycle).

The contraceptive effect was noticeably significant in one treatmentcycle (No. D06) when anti-E₂ levels were maintained for an extendedduration by repeated injections of E₂ -BSA. To further substantiate thatthe extended duration time of anti-E₂ levels capable of bindingapproximately 70 pg of E₂ or more per ml of serum causes an extendedcontraceptive effect, the following data (Table III) is shown.

                  TABLE III                                                       ______________________________________                                        Repeated Administration, Every 10 to 14 Days,                                 of Anti-E.sub.2 in 12 Female Dogs                                                       Control                Range of                                               (Untreated)    Treat-  Anti-E.sub.2 Titers                                    Estrous Cycle  ment    Throughout the                                         Lengths Prior  Cycle   Treatment                                              to Treatment   Length  Period                                       Dog No.   (Days)         (Days)  (PEA Units)                                  ______________________________________                                        7489          226              500   150-339                                                238                                                             9048          252              480+  103-408                                  16825         --               457+   98-378                                  12533         --               403    66-430                                  12889         --               600+  131-445                                  10221         234              239    80-413                                  3947          256              321    95-447                                                162                                                                           222                                                             4967          311              457    92-430                                                255                                                             6475          199              207   120-429                                                189                                                                           176                                                             8996          159              370    84-447                                                193                                                             1895          --                517+ 174-469                                  12090         229              306   132-453                                         Avg.   220         Avg. 405+                                           ______________________________________                                    

As shown in Table III, by administering repeated injections of anti-E₂to maintain titers sufficient to bind approximately 70 pg or more of E₂per ml of serum, one can definitely see a contraceptive effect asevidenced by the lengthened treatment cycle (avg. 405+ days) versus theaverage control cycle length (220 days). Although the anti-E₂ wasadministered passively, the results are still conclusive for the statedclaim.

To summarize briefly, this invention is directed to the discovery that avaccine which stimulates the production of antibodies against anestrogen which is essential to the reproductive cycle of a female mammalcan be prepared and used to sterilize the female. This has beengraphically illustrated by the use of female dogs as exemplary of themammals. The concept of the invention may, of course, be applied toother mammalian species such as humans, cows, horses, pigs, and thelike, it being necessary only to prepare an antigen which stimulates theproduction of antibodies against an estrogen which is essential to thereproductive cycle of the species. Although the concept is directed atblocking the reproductive cycle of the female, it is also contemplatedthat a vaccine which will stimulate the production of antibodies againsta necessary hormone to the reproductive process of the male may beprepared following the teaching of this invention.

In the selected species used herein for purposes of illustration of theinventive concept, the canine species, a vaccine which stimulates theproduction of antibodies to the estrogen, estradiol-17 beta, wasprepared. The estrogen was reacted with succinic anhydride to form thehemisuccinate and this derivative was coupled to Bovine Serum Albumin(BSA) to form a steriod-protein conjugate against which theimmunological system produces antibodies to E₂.

It was found that best results could be obtained if from 15 to 25molecules of the E₂ were coupled to each molecule of BSA. It is to berecognized, of course, that a much wider range than this is operable andit is contemplated that a carrier containing attached thereto from about5 to about 40 molecules of E₂ would cause the formation of a sufficientlevel of antibodies (titer) in the animal to be operable.

One important factor in obtaining satisfactory results with the vaccineof this invention was found to be the antibody level which was producedin the female. A contraceptive effect in female dogs utilizing theclaimed vaccine (E₂ -BSA) can be present if produced anti-E₂ titers aremaintained at approximately 70 PEA units or above to neutralizeendogenously produced E₂. It is to be understood, of course, that fordifferent species, different levels of estrogen antibodies may berequired. Functionally speaking, sufficient antibodies to effectivelyneutralize the estrogen levels required for a normal reproductive cyclemust be produced.

What is claimed is:
 1. A method of inducing a contraceptive effect in afemale canine having had at least one previous estrous cycle, whichmethod comprises parenterally administering to said female canine duringthe period subsequent to estrous, in an amount sufficient to bind atleast 70 pg of estradiol-17 beta per ml of serum, an injectablecomposition capable of preventing conception in a female canine, saidinjectable composition being produced by a process which comprises thesteps of: esterifying the hydroxyl groups of estradiol-17 beta withsuccinic anhydride to form the estradiol-17 beta hemisuccinate andcoupling said hemisuccinate to the amino group of a protein selectedfrom the group consisting of keyhole limpet hemocyanin, bovine serumalbumin and human serum albumin, about 5 to about 40 molecules ofestradiol-17 beta being bound to each molecule of the protein, to obtainan antigen and emulsifying said antigen in an adjuvant in the order of 1mg of antigen per 2 mls of adjuvant therefor.
 2. The method of claim 1wherein the protein is bovine serum albumin.
 3. The method of claim 2wherein 15-25 molecules of the estradiol-17 beta hemisuccinate arecoupled to each molecule of the bovine serum albumin.
 4. The method ofclaim 1 wherein the estradiol-17-beta succinate is coupled to theprotein via the carbodiimide condensation.